scholarly journals Optimized protocol for single-molecule RNA FISH to visualize gene expression in S. cerevisiae

2021 ◽  
Vol 2 (3) ◽  
pp. 100647
Author(s):  
Heta P. Patel ◽  
Ineke Brouwer ◽  
Tineke L. Lenstra
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Rna Fish ◽  
Optimized Protocol

scholarly journals Enzyme-based synthesis of single molecule RNA FISH probes

2018 ◽  
Author(s):  
Christian Lanctôt
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Low Cost ◽  
Single Cells ◽  
Rna Sequences ◽  
Rna Fish ◽  
Terminal Deoxynucleotide Transferase ◽  
Target Rna ◽  
Subcellular Level

ABSTRACTSingle molecule RNA fluorescence in situ hybridization (smRNA FISH) allows the quantitative analysis of gene expression in single cells. The technique relies on the use of pools of end-labeled fluorescent oligonucleotides to detect specific cellular RNA sequences. These fluorescent probes are currently chemically synthesized. Here I describe a novel technique based on the use of routine molecular biology enzymes to generate smRNA FISH probes without the need for chemical synthesis of pools of oligonucleotides. The protocol comprises 3 main steps: purification of phagemid-derived single stranded DNA molecules comprising a segment complementary to a target RNA sequence; fragmentation of these molecules by limited DNase I digestion; and end-labeling of the resulting oligonucleotides with terminal deoxynucleotide transferase and fluorescent dideoxynucleotides. smRNA FISH probes that are obtained using the technique presented here are shown to perform as well as conventional probes. The main advantages of the method are the low cost of probes and the flexibility it affords in the choice of labels. Enzyme-based synthesis of probes should further increase the popularity of smRNA FISH as a tool to investigate gene expression at the cellular or subcellular level.

4 Molecularly guided multiplexed digital spatial analysis reveals differential gene expression profiles in the WNT-β-catenin pathway between melanoma and prostate tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A4-A4
Author(s):  
Anushka Dikshit ◽  
Dan Zollinger ◽  
Karen Nguyen ◽  
Jill McKay-Fleisch ◽  
Kit Fuhrman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Single Molecule ◽  
Spatial Information ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Fluorescence Assay ◽  
Differentially Expressed ◽  
Tumor Type ◽  
Prostate Tumors

BackgroundThe canonical WNT-β-catenin signaling pathway is vital for development and tissue homeostasis but becomes strongly tumorigenic when dysregulated. and alter the transcriptional signature of a cell to promote malignant transformation. However, thorough characterization of these transcriptomic signatures has been challenging because traditional methods lack either spatial information, multiplexing, or sensitivity/specificity. To overcome these challenges, we developed a novel workflow combining the single molecule and single cell visualization capabilities of the RNAscope in situ hybridization (ISH) assay with the highly multiplexed spatial profiling capabilities of the GeoMx™ Digital Spatial Profiler (DSP) RNA assays. Using these methods, we sought to spatially profile and compare gene expression signatures of tumor niches with high and low CTNNB1 expression.MethodsAfter screening 120 tumor cores from multiple tumors for CTNNB1 expression by the RNAscope assay, we identified melanoma as the tumor type with the highest CTNNB1 expression while prostate tumors had the lowest expression. Using the RNAscope Multiplex Fluorescence assay we selected regions of high CTNNB1 expression within 3 melanoma tumors as well as regions with low CTNNB1 expression within 3 prostate tumors. These selected regions of interest (ROIs) were then transcriptionally profiled using the GeoMx DSP RNA assay for a set of 78 genes relevant in immuno-oncology. Target genes that were differentially expressed were further visualized and spatially assessed using the RNAscope Multiplex Fluorescence assay to confirm GeoMx DSP data with single cell resolution.ResultsThe GeoMx DSP analysis comparing the melanoma and prostate tumors revealed that they had significantly different gene expression profiles and many of these genes showed concordance with CTNNB1 expression. Furthermore, immunoregulatory targets such as ICOSLG, CTLA4, PDCD1 and ARG1, also demonstrated significant correlation with CTNNB1 expression. On validating selected targets using the RNAscope assay, we could distinctly visualize that they were not only highly expressed in melanoma compared to the prostate tumor, but their expression levels changed proportionally to that of CTNNB1 within the same tumors suggesting that these differentially expressed genes may be regulated by the WNT-β-catenin pathway.ConclusionsIn summary, by combining the RNAscope ISH assay and the GeoMx DSP RNA assay into one joint workflow we transcriptionally profiled regions of high and low CTNNB1 expression within melanoma and prostate tumors and identified genes potentially regulated by the WNT- β-catenin pathway. This novel workflow can be fully automated and is well suited for interrogating the tumor and stroma and their interactions.GeoMx Assays are for RESEARCH ONLY, not for diagnostics.

Reduction in gene expression noise by targeted increase in accessibility at gene loci

2021 ◽  
Vol 118 (42) ◽  
pp. e2018640118
Author(s):  
LaTasha C. R. Fraser ◽  
Ryan J. Dikdan ◽  
Supravat Dey ◽  
Abhyudai Singh ◽  
Sanjay Tyagi
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Single Molecule ◽  
Single Cells ◽  
Global Changes ◽  
Messenger Rnas ◽  
Histone Acetyl Transferase ◽  
Gene Expression Noise ◽  
Expression Noise ◽  
Eukaryotic Genes

Many eukaryotic genes are expressed in randomly initiated bursts that are punctuated by periods of quiescence. Here, we show that the intermittent access of the promoters to transcription factors through relatively impervious chromatin contributes to this “noisy” transcription. We tethered a nuclease-deficient Cas9 fused to a histone acetyl transferase at the promoters of two endogenous genes in HeLa cells. An assay for transposase-accessible chromatin using sequencing showed that the activity of the histone acetyl transferase altered the chromatin architecture locally without introducing global changes in the nucleus and rendered the targeted promoters constitutively accessible. We measured the gene expression variability from the gene loci by performing single-molecule fluorescence in situ hybridization against mature messenger RNAs (mRNAs) and by imaging nascent mRNA molecules present at active gene loci in single cells. Because of the increased accessibility of the promoter to transcription factors, the transcription from two genes became less noisy, even when the average levels of expression did not change. In addition to providing evidence for chromatin accessibility as a determinant of the noise in gene expression, our study offers a mechanism for controlling gene expression noise which is otherwise unavoidable.

scholarly journals Spatially resolved transcriptomics reveals unique gene signatures associated with human temporal cortical architecture and Alzheimer's pathology

2021 ◽  
Author(s):  
Shuo Chen ◽  
Yuzhou Chang ◽  
Liangping Li ◽  
Diana Acosta ◽  
Cody Morrison ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Brain Region ◽  
Expression Patterns ◽  
Middle Temporal Gyrus ◽  
Marker Genes ◽  
Gene Signatures ◽  
Unique Gene ◽  
Spatially Resolved ◽  
And Gender

Alzheimer's disease (AD) is pathologically characterized by amyloid beta (Aβ) plaques, neurofibrillary tangles (tau aggregates), and alterations in microglia, astrocytes and oligodendrocytes. The mesial temporal lobe is a vulnerable brain region in early AD; however, little is known about the transcriptome-scale gene expression in this region and its relation to AD pathology. Here we use the 10x Genomics Visium platform in combination with co-immunofluorescence staining of AD-associated pathological markers to define the spatial topography of gene expression in the middle temporal gyrus (MTG) from both early AD and age- and gender-matched control cases. We identify unique marker genes for six cortical layers and the adjacent white matter as well as gene expression patterns and alterations that showcase unique gene signatures and pathways associated with a range of AD pathology. Also, gene co-expression analyses of differentially expressed genes (DEGs) between AD and controls reveal four unique gene modules, which significantly change their co-expression patterns in the presence of variations of AD pathology. Furthermore, we validate the changes of key representative DEGs that are associated with AD pathology in neurons, microglia, astrocytes and oligodendrocytes using single-molecule fluorescent in situ hybridization. In summary, we provide a rich resource for the spatial transcriptomic profile of the human MTG, which will contribute to our understanding of the complex architecture and AD pathology of this vulnerable brain region.

scholarly journals Transcription factor residence time dominates over concentration in transcription activation

2020 ◽  
Author(s):  
Achim P. Popp ◽  
Johannes Hettich ◽  
J. Christof M. Gebhardt
Keyword(s):  
Transcription Factor ◽  
Residence Time ◽  
Single Molecule ◽  
Search Time ◽  
Mrna Synthesis ◽  
Burst Frequency ◽  
Quantitative Measurements ◽  
Rna Fish ◽  
Transition Times ◽  
Basic Course

Transcription is a vital process activated by transcription factor (TF) binding. The active gene releases a burst of transcripts before turning inactive again. While the basic course of transcription is well understood, it is unclear how binding of a TF affects the frequency, duration and size of a transcriptional burst. We systematically varied the residence time and concentration of a synthetic TF and characterized the transcription of a reporter gene by combining single molecule imaging, single molecule RNA-FISH, live transcript visualisation and analysis with a novel algorithm, Burst Inference from mRNA Distributions (BIRD). For this well-defined system, we found that TF binding solely affected burst frequency and variations in TF residence time had a stronger influence than variations in concentration. This enabled us to device a model of gene transcription, in which TF binding triggers multiple successive steps before the gene transits to the active state and actual mRNA synthesis is decoupled from TF presence. We quantified all transition times of the TF and the gene, including the TF search time and the delay between TF binding and the onset of transcription. Our quantitative measurements and analysis revealed detailed kinetic insight, which may serve as basis for a bottom-up understanding of gene regulation.

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